Biomarkers for early diagnosis and differentiation of mycobacterial infection

ABSTRACT

Mycobacterial-specific biomarkers and methods of using such biomarkers for diagnosis of mycobacterial infection in a mammal are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/069,520, filed Oct.28, 2014 and incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 58-3148-0-174awarded by the USDA/ARS and 2012-33610-19517 awarded by the USDA/NIFA.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The field of the invention is vaccine and diagnostic biomarkers. Moreparticularly, the invention relates to a set of biomolecules asdiagnostic biomarkers for distinguishing between vaccinated and infectedanimals and between M. ap. and M. bovis.

Mycobacterial infections cause significant health problems to humans andanimals including human tuberculosis, bovine tuberculosis, and Johne'sdisease. Johne's disease (aka paratuberculosis) is caused by infectionwith Mycobacterium avium subspecies paratuberculosis (M. ap); thisdisease causes severe economic losses estimated at $500 million per yearfor the US dairy industry alone, and these infections constitute aproblem for 91% of dairy herds. Bovine tuberculosis, which is caused byinfection with M. bovis, is endemic in dairy herds in several parts ofthe developing world and a significant problem for the wildlife animalsin several developed countries (e.g., UK, USA, and Australia).

Current diagnostics can detect mycobacterial infections in cattle thathave started to shed the bacteria or developed an antibody response. Theavailable diagnostic tools are unreliable to detect early stages ofinfection or to differentiate infected from vaccinated animals (aka theDIVA principle). Early detection of mycobacterial infections isimperative to control the infection in herds. Further, the availabilityof a DIVA-based assay will facilitate adoption of new vaccines that canprevent M. ap infection.

Needed in the art are methods or diagnostic tools for detecting earlystages of mycobacterial infection. Additionally, needed in the art aremethods or diagnostic tools for distinguishing vaccinated from infectedanimals and distinguishing M. ap. from M. bovis pathogens in infectedanimals.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding a set of pathogen and animal biomolecules for detectingmycobacterial infections in the early stage. By using the set ofbiomolecules as biomarkers, one can distinguish between vaccinated andinfected animals, distinguish between M. ap. and M. bovis pathogens ininfected animals, and distinguish M. ap. and M. bovis infections.

In one aspect, the present invention discloses a method for diagnosis ofmycobacterial infection in a mammal The method comprises the steps of(a) obtaining a sample from the mammal; (b) testing the sample for theconcentration level of at least one mycobacterial-specific biomarker andcomparing the level of the biomarker against the level detected in anuninfected mammalian sample; and (c) determining the infection status ofthe mammal.

In one embodiment, the method is used for early diagnosis and detectionof mycobacterial infection in a mammal In one embodiment, the testing isvia ELISA assay for antibodies formed against the biomarker. In oneembodiment, the sample is a blood sample.

In one embodiment, the mammal of the present invention is selected fromthe group consisting of bubaline, elephantine, musteline, pardine,phocine, rhinocerine, caprine, hircine, leonine, leporine, lupine,lyncine, murine, rusine, tigrine, ursine, vulpine, zebrine,vespertilionine, porcine, bovine, equine, swine, elaphine, ovine,caprine, camelidae, feline, cervine, primate, human and canine mammals.In one embodiment, the mammal is selected from the group of pig, cow,human, rodent, sheep, goat and deer. In one embodiment, the mammal isselected from the group consisting of cow, sheep and goat. In onespecific embodiment, the mammal is a cow.

In one embodiment, the mycobacterial-specific biomarker used in thepresent method comprises at least one member selected from the groupconsisting of gene sequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6,Q73XZO, Q740D1 and Q73UE0 and expression products derived thereof.

In one embodiment, the mycobacterial-specific biomarker used in thepresent method comprises at least one member selected from the groupconsisting of gene sequences Q73VL6, Q73YW9, Q741L4, Q744E5, Q 73YP5,Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 and expressionproducts derived thereof.

In another embodiment, the present invention comprises host biomarkerslisted in Table 3.

In one embodiment, the biomarker used in the present invention comprisesa protein having at least 50%, 60%, 70%, 80%, or 90%_of the amino acidsequence of M. paratuberculosis which is not conserved in M. bovis, asexemplified by the amino acid difference between M. paratuberculosis andM. bovis in FIG. 1.

In one aspect, the present invention discloses a method fordifferentiating a vaccinated mammal from a non-vaccinated mammal or froman infected mammals, the method comprising the steps of (a) obtaining asample from the test mammal; (b) testing the sample for theconcentration level of at least one of mycobacterial specific markersand comparing the level of the markers with that detected in anuninfected animal; and (c) determining the status of the mammal.

In one embodiment, the mammals are vaccinated with a mycobacteriummutant vaccine.

In one embodiment, the mycobacterium mutant vaccine comprises at leastone mutation in at least one gene sequence encoding global generegulators (GGRs) selected from the group consisting of sigH, sigL andLipN.

In one embodiment, the marker comprises at least one member selectedfrom the group consisting of Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6,Q73XZO, Q740D1 and Q73UE0 and expression products derived thereof.

In one embodiment, the marker comprises at least one member selectedfrom the group consisting of Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5,Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 and expressionproducts derived thereof.

In one embodiment, the marker comprises a protein having at least 50%,60%, 70%, 80%, or 90% of the amino acid sequence of M. paratuberculosiswhich is not conserved in M. bovis as exemplified by the amino aciddifference between M. paratuberculosis and M. bovis in FIG. 1.

In one aspect, the present invention discloses a method fordifferentiating a M. ap infected mammal from a M. bovis infected mammal,the method comprising the steps of (a) obtaining a sample from the testmammal; (b) testing the sample for the concentration level of at leastone of mycobacterial-specific markers and comparing the level of themarkers with that detected in a M. bovis infected mammal; and (c)determining the status of the mammal.

In one embodiment, the mycobacterial-specific markers comprise a proteinhaving at least 50%, 60%, 70%, 80%, or 90% of the amino acid sequence ofM. paratuberculosis which is not conserved in M. bovis.

In one aspect, the present invention discloses a set of biomarkers forearly diagnosis and differentiation of mycobacterial infection. Thebiomarkers comprise at least one member selected from the groupconsisting of Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1 andQ73UE0 and expression products derived thereof.

In one embodiment, the biomarkers comprise at least two members selectedfrom the group consisting of Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6,Q73XZ0, Q740D1 and Q73UE0 and expression products derived thereof.

In one aspect, the present invention discloses set of biomarkers forearly diagnosis and differentiation of mycobacterial infection. Thebiomarkers comprise at least one member selected from the groupconsisting of Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21,Q73UH9, Q741M5, Q742F4, and Q73SU6 and encoded genes or expressionproducts derived thereof.

In one embodiment, the biomarkers comprise at least two members selectedfrom the group consisting of Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5,Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 and encoded genes orexpression products derived thereof.

In one aspect, the present invention discloses a set of biomarkers forearly diagnosis and differentiation of mycobacterial infection. Thebiomarkers comprise a protein having at least 50%, 60%, 70%, 80%, 90%,95% or 100% of the amino acid sequence of M. paratuberculosis which isnot conserved in M. bovis.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, reference is made therefore to the claims andherein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

FIG. 1 is a graph showing the alignment plot of amino acids deduced fromthe protein sequence in LipN of both M. paratuberculosis and M. bovis.Peptides conserved in M. paratuberculosis sequence but absent from M.bovis are the target for developing the DIVA test.

FIG. 2A is a diagram showing multiplex PCR strategy using 3 primers,specifically, Wild-type (virulent) strain with intact lipN gene.

FIG. 2B is a diagram showing multiplex PCR strategy using 3 primers,specifically, LAV strain with scar sequence from hygromycin cassetteremoval represented by the black rectangle.

DETAILED DESCRIPTION OF THE INVENTION

The term “mycobacterium,” as used herein, refers to a genus ofactinobacteria given its own family, the mycobacteriaceae. The genusincludes pathogens known to cause serious diseases in mammals, includingtuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacteriumleprae).

Mycobacterium tuberculosis complex (MTBC) members are causative agentsof human and animal tuberculosis. Species in this complex may include M.tuberculosis, the major cause of human tuberculosis, M. bovis, M. bovisBCG, M. africanum, M. canetti, M. caprae, M. microti, and M. pinnipedii.

Mycobacterium avium complex (MAC) is a group of species that, in adisseminated infection but not lung infection, used to be a significantcause of death in AIDS patients. Species in this complex include M.avium, M. avium paratuberculosis, which has been implicated in Crohn'sdisease in humans and is the causative agent of Johne's disease incattle and sheep, M. avium silvaticum, M. avium “hominissuis,” M.colombiense, and M. indicus pranii.

Mycobacterial infections are notoriously difficult to treat. Theorganisms are hardy due to their cell wall, which is neither truly Gramnegative nor positive. In addition, they are naturally resistant to anumber of antibiotics that disrupt cell-wall biosynthesis, such aspenicillin. Due to their unique cell wall, they can survive longexposure to acids, alkalis, detergents, oxidative bursts, lysis bycomplement, and many antibiotics. Most mycobacteria are susceptible tothe antibiotics clarithromycin and rifamycin, but antibiotic-resistantstrains have emerged.

The term “biomolecule,” as used herein, refers to any organic moleculethat is part of or from a living organism. Biomolecules may includenucleic acids, a nucleotide, a polynucleotide, an oligonucleotide, apeptide, a protein, a carbohydrate, a ligand, a receptor, among others.In one embodiment of the present invention, biomolecules may includegenes and their expression products.

The term “expression product,” as used herein, refers to any productproduced during the process of gene expression. These products are oftenproteins, but in non-protein coding genes such as ribosomal RNA (rRNA),transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is afunctional RNA.

The terms “polypeptide,” “peptide,” and “protein,” as used herein, referto a polymer comprising amino acid residues predominantly bound togetherby covalent amide bonds. The terms apply to amino acid polymers in whichone or more amino acid residue may be an artificial chemical mimetic ofa naturally occurring amino acid, as well as to naturally occurringamino acid polymers and non-naturally occurring amino acid polymers. Asused herein, the terms may encompass amino acid chains of any length,including full length proteins, wherein the amino acids are linked bycovalent peptide bonds. The protein or peptide may be isolated from anative organism, produced by recombinant techniques, or produced bysynthetic production techniques known to one skilled in the art.

The term “recombinant protein,” as used herein, refers to a polypeptideof the present disclosure which is produced by recombinant DNAtechniques, wherein generally, DNA encoding a polypeptide is insertedinto a suitable expression vector which is in turn used to transform aheterologous host cell (e.g., a microorganism or yeast cell) to producethe heterologous protein.

The term “recombinant nucleic acid” or “recombinant DNA,” as usedherein, refers to a nucleic acid or DNA of the present disclosure whichis produced by recombinant DNA techniques, wherein generally, DNAencoding a polypeptide is inserted into a suitable expression vectorwhich is in turn used to transform a host cell to produce theheterologous protein.

The term “mammal,” as used herein, refers to any living species whichcan be identified by the presence of sweat glands, including those thatare specialized to produce milk to nourish their young. In oneembodiment, the mammal suitable for the present invention may includebubaline, elephantine, musteline, pardine, phocine, rhinocerine,caprine, hircine, leonine, leporine, lupine, lyncine, murine, rusine,tigrine, ursine, vulpine, zebrine, vespertilionine, porcine, bovine,equine, swine, elaphine, ovine, caprine, camelidae, feline, cervine,primate, human and canine mammals. In one preferred embodiment of thepresent invention, the mammal may be one of the ruminants such ascattle, goats, sheep, giraffes, yaks, deer, camels, llamas, antelope,and some macropods. In one specific embodiment of the present invention,the mammal may include any of the milk cattle species, such as cow,sheep and goat.

The term “antibody,” as used herein, refers to a class of proteins thatare generally known as immunoglobulins. The term “antibody” herein isused in the broadest sense and specifically includes full-lengthmonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments, so long as theyexhibit the desired biological activity. Various techniques relevant tothe production of antibodies are provided in, e.g., Harlow, et al.,ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

The term “marker” or “biomarker,” as used herein, refers to abiomolecule (e.g., protein, nucleic acid, carbohydrate, or lipid) thatis differentially expressed in the cell, differentially expressed on thesurface of an infected cell, differentially phosphorylated, ordifferentially secreted by a infected cell in comparison to a normalcell or in a paracrine fashion by neighboring uninfected cells, andwhich is useful for the diagnosis of mycobacterial infection and forpreferential targeting of a pharmacological agent to an infected mammalOften times, such markers are molecules that are over-expressed in aninfected cell in comparison to a normal cell, for instance, at least1-fold over-expression, at least 2-fold over-expression, at least 3-foldover-expression or more in comparison to a normal cell.

The term “mycobacterial-specific biomarkers,” as used herein, refers tobiomarkers which are specifically related to mycobacterial infection.Some of these biomarkers are listed at FIG. 1 and Tables 1 and 2.

The term “lyophilization,” as used herein, refers to freezing of amaterial at low temperature followed by dehydration by sublimation,usually under a high vacuum. Lyophilization is also known as freezedrying. Many techniques of freezing are known in the art oflyophilization such as tray freezing, shelf freezing, spray-freezing,shell-freezing and liquid nitrogen immersion. Each technique will resultin a different rate of freezing. Shell freezing may be automated ormanual. For example, flasks can be automatically rotated by motor drivenrollers in a refrigerated bath containing alcohol, acetone, liquidnitrogen, or any other appropriate fluid. A thin coating of product isevenly frozen around the inside “shell” of a flask, permitting a greatervolume of material to be safely processed during each freeze drying run.Tray freezing may be performed by, for example, placing the samples inlyophilizer, equilibrating 1 hr at a shelf temperature of 0° C., thencooling the shelves at 0.5° C./min to −40° C. Spray-freezing, forexample, may be performed by spray freezing into liquid, dropping by ˜20μl droplets into liquid N₂, spray freezing into vapor over liquid, or byother techniques known in the art.

The term “antigen,” as used herein, refers to any molecule that iscapable of eliciting an immune response, whether a cell-mediated orhumoral immune response, whether in the presence or absence of anadjuvant. An antigen can be any type of molecule, e.g., a peptide orprotein, a nucleic acid, a carbohydrate, a lipid, and combinationsthereof. A “vaccine antigen” is an antigen that can be used in a vaccinepreparation. A “therapeutic antigen” is an antigen that can be used fortherapeutic purposes.

The term “vaccine,” as used herein, refers to an antigenic preparationused to produce active immunity to a disease, in order to prevent orameliorate the effects of infection. The antigenic moiety making up thevaccine may be either a live or killed microorganism, or a naturalproduct purified from a microorganism or other cell including, but notlimited to tumor cells, a synthetic product, a genetically engineeredprotein, peptide, polysaccharide or similar product or an allergen.

The term “immunologically active,” as used herein, refers to the abilityto raise one or more of a humoral response or a cell mediated responsespecific to an antigen.

The term “adjuvant,” as used herein, refer to compounds that, when usedin combination with specific vaccine antigens in formulations, augmentor otherwise alter or modify the resultant immune responses. An adjuvantcombined with a vaccine antigen increases the immune response to thevaccine antigen over that induced by the vaccine antigen alone. Anadjuvant may augment humoral immune responses or cell-mediated immuneresponses or both humoral and cell-mediated immune responses againstvaccine antigens.

The term “detecting,” as used herein, refers to confirming the presenceof the biomarker or marker present in the sample. Quantifying the amountof the biomarker or marker present in a sample may include determiningthe concentration of the biomarker present in the sample. Detectingand/or quantifying may be performed directly on the sample, orindirectly on an extract therefrom, or on a dilution thereof.

In one aspect, the present invention relates to a method for diagnosisof mycobacterial infection in a mammal In one embodiment, the presentinvention discloses a method for early detection of mycobacterialinfection. The term “early detection,” as used herein, refers todetection of mycobacterial infection during the early stage ofinfection, e.g., a stage before the development of chronic diarrhea.

In another embodiment, the present invention discloses a method fordifferentiating a vaccinated mammal from a non-infected mammal or amycobacterial infected mammal.

In another embodiment, the present invention discloses a method fordifferentiating mammals infected by one kind of mycobacteria fromanother kind of mycobacteria.

In one specific embodiment, the present invention discloses a method fordifferentiating a M. ap infected mammal from a M. bovis infected mammal

The detection of mycobacterial infection and related diseases such asJohne's disease is very difficult because the disease generally takesmany years to develop, and the organism is shed by the mammalperiodically, so every mammal must be repeatedly tested over long timeperiods.

Applicants have identified mycobacterial-specific biomarkers, such asgenes and/or expression products derived thereof, useful for detectionof mycobacterial infection. Mycobacterial-specific biomarkers or acombination of such biomarkers may also be used to differentiate avaccinated mammal (e.g., from genetically engineered vaccines) from anon-infected mammal or a mycobacterial-infected mammal. Further,mycobacterial-specific biomarkers or a combination of such biomarkersmay also be used to differentiate one pathogen (e.g., M.paratuberculosis) from another pathogen (e.g., M. bovis) for infectedmammals.

Differentiating Vaccinated Mammals from Mycobacterial-Infected Mammals

In one embodiment, the present invention discloses a method fordifferentiating a vaccinated mammal from a mycobacterial infected mammal

In one embodiment, the method for differentiating a vaccinated mammalfrom a mycobacterial-infected mammal comprises the steps of (a)obtaining a sample from the mammal; (b) testing the sample for theconcentration level of at least one mycobacterial-specific biomarker andcomparing the level of the biomarker against the level detected in anuninfected mammalian sample; and (c) determining the infection status ofthe mammal.

A sample suitable for the present invention may include any biologicalsample from the mammal The biological sample may include, withoutlimitation, saliva, sputum, blood, plasma, serum, urine, feces,cerebrospinal fluid, amniotic fluid, wound exudate, or tissue of thesubject of mammal In one specific embodiment, the biological sample is ablood sample.

A major problem in employing mass vaccination program for the control ofJD in dairy herds is the inability to differentiate between infected andvaccinated animals with the current vaccine (DIVA principal). Applicantshave previously proposed using genetically engineered vaccines (PCTpatent application US2014/02024). One would wish to consider the DIVAprincipal and wish to distinguish between M. bovis and JD vaccinatedanimals that have been vaccinated with genetically engineered vaccines.

In one embodiment, Applicants identify biomolecules asmycobacterial-specific biomarkers. For example, the biomolecules ofmycobacterial-specific biomarkers may include genes and their expressionproducts which are present in a M. ap wild-type strain but not presentor have a low expression level in genetically engineered vaccines.

In another embodiment, the present invention comprises host biomarkerslisted in Table 3.

Applicants envision that the present invention may be applicable to anygenetically engineered vaccines. In one specific embodiment, the presentinvention is applicable to live attenuated vaccines. The example of thelive attenuated vaccines may include sigL and sigH mutants. PCT patentapplication US2014/020248 discloses live attenuated vaccines, such assigL and sigH mutants.

In one embodiment involving sigL and sigH mutants, themycobacterial-specific biomarker may comprise at least one memberselected from the group consisting of gene sequences Q73SF4, Q73Y73,Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1 and Q73UE0 and expressionproducts derived thereof. In another embodiment, themycobacterial-specific biomarker may comprise at least two, three, four,five, six, seven or eight members selected from the group consisting ofgene sequences Q73 SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1and Q73UE0 and expression products derived thereof. Preferably, themycobacterial-specific biomarker may comprise at least two membersselected from the group as discussed above.

In one embodiment involving sigL and sigH mutants, themycobacterial-specific biomarker comprises at least one member selectedfrom the group consisting of gene sequences Q73VL6, Q73YW9, Q741L4,Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 andexpression products derived thereof.

In one embodiment, the mycobacterial-specific biomarker comprises atleast two, three, four, five, six, seven, eight, nine or ten membersselected from the group consisting of gene sequences Q73VL6, Q73YW9,Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, andQ73SU6 and expression products derived thereof.

In one embodiment, the mycobacterial-specific biomarker comprises allgene sequences Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21,Q73UH9, Q741M5, Q742F4, and Q73SU6 and expression products derivedthereof.

In one embodiment, the presence or absence of the biomarkers in a mammalmay demonstrate the infection status of the mammal In one specificembodiment, the biomarkers that are significantly over-expressed in thewild type strain and not in the mutant vaccine and could be used for themutant vaccine-DIVA testing.

For example, when the biomarkers are those significantly over-expressedin animals infected with the wild type strain and not in the mutantvaccine, the presence of at least one biomarker in a mammal shows thatthe mammal may be infected and not merely vaccinated. On the other hand,the absence of at least one biomarker in a mammal shows that the mammalmay be vaccinated.

In one embodiment, Applicants envision that the present invention isalso applicable when antigens are inoculated to a mammal and theinfection status of the mammal needs to be identified. Specifically, theinfection status may include whether a mammal is vaccinated or whether amammal is infected with M. paratuberculosis or M. bovis.

In one embodiment, Applicants envision that the present invention mayalso be used in a skin test. For example, Applicants envision that amammal may be inoculated with a reagent comprising any suitable antigenor a biomarker as discussed herein, and skin induration of the mammalmay be recorded. Skin induration may be used to differentiate infectedfrom vaccinated animals.

Early Stage Detection Methods

In another embodiment, the concentration level of the biomarkers may becompared with the level detected in a standard sample, such as anuninfected mammalian sample or those infected for long periods of time(chronic infection). The concentration level of the biomarkers in amammalian sample may demonstrate the infection status of the mammal Forexample, a low concentration level of the biomarkers may indicate thatthe mammal is in a early stage of infection.

In one specific embodiment, a biomarker having at least one gene or acombination of at least two genes from the group consisting of genesequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1 andQ73UE0 and expression products thereof may demonstrate the infectionstatus of a mammal related to sigL-based vaccines. In another specificembodiment, a biomarker having at least one gene or a combination of atleast two genes from the group consisting of gene sequences Q73VL6,Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4,and Q73 SU6 may demonstrate the infection status of a mammal related tosigH-based vaccines. In another embodiment, the present inventioncomprises host biomarkers listed in Table 3.

Further, the expression level of the biomarkers and/or specificcombination of the biomarkers may allow early detection of mycobacterialinfection. In one specific embodiment, the biomarkers may include atleast one gene or a combination of at least two genes from the groupconsisting of gene sequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6,Q73XZO, Q740D1 and Q73UE0 and expression products thereof In anotherspecific embodiment, the biomarkers may include at least one gene or acombination of at least two genes from the group consisting of genesequences Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21,Q73UH9, Q741M5, Q742F4, and Q73SU6 and expression products thereof.

Applicants envision that the mycobacterial-specific biomarker mayinclude genes or the polynucleotides containing less than an entiremicrobial or mammalian species genomes of the above genes. The biomarkerof genes or the polynucleotides may be either single-or double-strandednucleic acids. A polynucleotide may be RNA, DNA, cDNA, genomic DNA,chemically synthesized RNA or DNA or combinations thereof. Thepolynucleotides can be purified free of other components, such asproteins, lipids and other polynucleotides. For example, thepolynucleotide may be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%purified. The purified polynucleotides may comprise additionalheterologous nucleotides (i.e., nucleotides that are not frommycobacterium). The purified polynucleotides of the invention can alsocomprise other nucleotide sequences, such as sequences coding forlinkers, primer, signal sequences, TMR stop transfer sequences,transmembrane domains, or ligands.

The gene or the polynucleotides of the invention may also comprisefragments that encode immunogenic polypeptides. Polynucleotides of theinvention may encode full-length polypeptides, polypeptide fragments,and variant or fusion polypeptides. Polynucleotides of the invention maycomprise coding sequences for naturally occurring polypeptides or mayencode altered sequences that do not occur in nature. If desired,polynucleotides may be cloned into an expression vector comprisingexpression control elements, including for example, origins ofreplication, promoters, enhancers, or other regulatory elements thatdrive expression of the polynucleotides of the invention in host cells.

Differentiating M. ap-Infected Mammals from M. bovis-Infected Mammals

In another aspect, the present invention discloses a method fordifferentiating a M. ap-infected mammal from a M. bovis-infected mammalThe method comprises the steps of (a) obtaining a sample from the testmammal; (b) testing the sample for the concentration level of at leastone of mycobacterial-specific markers and comparing the level of themarkers with that detected in a M. bovis infected mammal; and (c)determining the status of the mammal.

In one embodiment, Applicants envision that the present invention mayalso be used in a skin test. For example, Applicants envision that amammal may be inoculated with a reagent comprising any suitable antigenor a biomarker as discussed herein, and characteristic symptom of themammal (e.g., skin induration, redness, size, and others) may berecorded. One can compare the characteristic symptom of the mammal withthose in a standard (an infected mammal, non-infected mammal, M.ap-infected mammal or M. bovis-infected mammal) to determine theinfection status of the mammal. Specifically, the present method may beused to differentiate infected from vaccinated animals.

In one embodiment, Applicants identify proteins or polypeptides asmycobacterial-specific markers for differentiating a M. ap infectedmammal from a M. bovis infected mammal. Specifically, themycobacterial-specific markers may comprise peptides conserved in M.paratuberculosis sequence but absent from M. bovis. Alternatively, themycobacterial-specific markers may comprise peptides conserved in M.bovis sequence but absent from M. paratuberculosis sequence.

FIG. 1 demonstrates the alignment plot of amino acids deduced from theprotein sequence in LipN of both M. paratuberculosis and M. bovis. Inone embodiment, the biomarker comprises a protein or peptide sequencehaving at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the amino acidsequence of M. paratuberculosis which is not conserved in M. bovis (seethe amino acid difference between M. paratuberculosis and M. bovis inFIG. 1) over a length of 2-100, 4-50, 6-30, and preferably 8-20 aminoacids. In one embodiment, Applicants envision that a length of 8-20amino acids that differentiate between M. paratuberculosis and M. bovis,as shown in FIG. 1, may be sufficient to differential between theinfected of the two strains (M. ap and M. bovis).

In one embodiment, the concentration level of at least one of themycobacterial-specific markers may be compared with that detected in aM. bovis infected mammal For example, the presence (e.g., having adetectable concentration level) of the biomarkers specific for M.paratuberculosis shows the mammal may be M. ap infected. Theconcentration level as compared with a standard sample may also provideinformation regarding infection status such as early infection.

Detection of Biomarkers or Markers

The present biomarkers or markers may be detected by any suitablemethod. In one embodiment, the testing is via ELISA assay for antibodiesformed against the biomarkers or markers.

The biomarker or marker in the present invention may be directlydetected, e.g., by SELDI or MALDI-TOF. Alternatively, the biomarker maybe detected directly or indirectly via interaction with a ligand orligands such as an antibody or a biomarker-binding fragment thereof, orother peptide, or ligand, e.g. aptamer, or oligonucleotide, capable ofspecifically binding the biomarker. The ligand may possess a detectablelabel, such as a luminescent, fluorescent or radioactive label, and/oran affinity tag.

For example, detecting and/or quantifying may be performed by one ormore method(s) selected from the group consisting of: SELDI (-TOF),MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Massspectrometry (MS), reverse phase (RP) LC, size permeation (gelfiltration), ion exchange, affinity, HPLC, UPLC and other LC or LCMS-based techniques. Appropriate LC MS techniques may include ICAT®(Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA).Liquid chromatography (e.g., high pressure liquid chromatography (HPLC)or low pressure liquid chromatography (LPLC)), thin-layerchromatography, NMR (nuclear magnetic resonance) spectroscopy may alsobe used. Methods of diagnosing and/or monitoring according to theinvention may comprise analyzing a plasma, serum or whole blood sampleby a sandwich immunoassay to detect the presence or level of thebiomarker. These methods are also suitable for clinical screening,prognosis, monitoring the results of therapy, identifying patients mostlikely to respond to a particular therapeutic treatment, for drugscreening and development, and identification of new targets for drugtreatment.

Detecting and/or quantifying the biomarkers or markers may be performedusing an immunological method, involving an antibody, or a fragmentthereof capable of specific binding to the biomarker. Suitableimmunological methods include sandwich immunoassays, such as sandwichELISA, in which the detection of the analyte biomarkers is performedusing two antibodies which recognize different epitopes on a analytebiomarker; radioimmunoassays (RIA), direct, indirect or competitiveenzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA),Fluorescence immunoassays (FIA), western blotting, immunoprecipitationand any particle-based immunoassay (e.g., using gold, silver, or latexparticles, magnetic particles, or Q-dots) Immunological methods may beperformed, for example, in microtiter plate or strip format.

The gene or the polynucleotides of the invention may be detected by, forexample, a probe or primer or a PCR primer. The gene or thepolynucleotides of the invention may be the basis for designing acomplimentary probe or primer, to detect the presence and/or quantity ofmycobacterium in a subject, such as a biological sample. Probes aremolecules capable of interacting with a target nucleic acid, typicallyin a sequence specific manner, for example, through hybridization.Primers are a subset of probes that can support an specific enzymaticmanipulation and that can hybridize with a target nucleic acid such thatthe enzymatic manipulation occurs. A primer may be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art that do not interfere with the enzymaticmanipulation. “Specific” means that a gene sequence recognizes ormatches another gene of the invention with greater affinity than toother non-specific molecules. Preferably, “specifically binds” or“specific to” also means a gene sequence recognizes and matches a genesequence comprised in a wild-type mycobacterium or a mycobacteriummutant described herein, with greater affinity than to othernon-specific molecules. More preferably, the probe or the primer iscomplimentary to a mycobacterium mutant with at least one mutation inthe gene, e.g., sigH or sigL.

The hybridization of nucleic acids is well understood in the art.Typically a primer may be made from any combination of nucleotides ornucleotide derivatives or analogs available in the art. The ability ofsuch primers to specifically hybridize to mycobacterium polynucleotidesequences will enable them to be of use in detecting the presence ofcomplementary sequences in a given subject. The primers of the inventionmay hybridize to complementary sequences in a subject such as abiological sample, including, without limitation, saliva, sputum, blood,plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, woundexudate, or tissue of the subject. Polynucleotides from the sample canbe, for example, subjected to gel electrophoresis or other sizeseparation techniques or can be immobilized without size separation.

The probes or the primers may also be labeled for the detection.Suitable labels, and methods for labeling primers are known in the art.For example, the label may include, without limitation, radioactivelabels, biotin labels, fluorescent labels, chemiluminescent labels,bioluminescent labels, metal chelator labels and enzyme labels. Thepolynucleotides from the sample are contacted with the probes or primersunder hybridization conditions of suitable stringencies. Preferably, theprimer is fluorescent labeled. Also, the detection of the presence orquality of the gene sequence of interest can be accomplished by anymethod known in the art. For instance, the detection can be made by aDNA amplification reaction. In some embodiments, “amplification” of DNAdenotes the use of polymerase chain reaction (PCR) to increase theconcentration of a particular DNA sequence within a mixtures of DNAsequences.

In another embodiment, the amplification of DNA may be done by theloop-mediated isothermal amplification (LAMP). Similar to PCR, LAMPutilizes a polymerization-based reaction to amplify DNA from examinedsamples, but the enzyme for LAMP, Bst DNA polymerase large fragment,possesses a DNA strand displacement activity. This makes the DNAextension step possible without having to fully denature DNA templates.Moreover, the primers are designed in a way that a hairpin loopstructure is formed in the first cycle of amplification, and thefollowing products are further amplified in an auto-cycling manner.Therefore, in about an hour, the repeated reactions can amplify by ˜10⁹copies of DNA molecules and can be done at a constant temperature in asingle heat block, instead of at various cycles of temperature in arelatively expensive thermal cycler. The detection of LAMP has beendescribed in PCT patent application US2014/020248.

In one embodiment, the detection of the presence of the gene or thespecific binding between the gene in mycobacterium mutant and a genethat is not a component of a subject's immune response to a particularvaccine may indicate a natural or experimental mycobacterium infection.For example, the absence of such binding or presence may indicate theabsence of mycobacterium infection. Or, a second, separate gene, such asa mutated mycobacterium gene that is specific to a component of amammal's immune response to a particular mycobacterium vaccine, may beused to detect corresponding antibodies produced in response tovaccination. Thus, if an antibody specific to a gene in mycobacteriumvaccine is detected, then the mammal has been vaccinated and/orinfected. The detection of neither genes indicates no infection and novaccination. As such, various combinations can lead to a determinationof the vaccination and/or infection status of the mammal

Additional Markers

Table 3, which tabulates the result of one of the Examples drawn to hosttranscriptome analysis of goats, lists additional markers that will beuseful for embodiments of the invention. The first part of Table 3 listsDNA markers that are useful for early diagnosis of John's disease inruminants, as described above, because the markers differentiateinfected from naive animals. The Table lists the locus in goats andprovides homologous locus in cows, if it is known. Table 3 also listshost markers that can differentiate live attenuated vaccinated animalsfrom naive animals and markers that can differentiateinactivated-vaccine immunized from naive animals.

Kits of the Present Invention

In another aspect, the present invention discloses a diagnostic kitsuitable for carrying out the diagnostic method of the first aspect ofthe invention. In one embodiment, the kit may be a “one-day” kit,meaning that it is capable of providing the diagnostic result within oneday of sample collection. In another embodiment, the kit may be able toprovide a diagnostic result within 12, 10, 8, 6, 4, 2, 1 or 0.5 hours ofsample collection.

In one embodiment, the diagnostic kit may be used for early detection ofmycobacterial infection in a mammal. The diagnostic kit may also be usedto differentiate a vaccinated mammal from an infected mammal. Forinfected mammals, the diagnostic kit may further be used todifferentiated different pathogens such as M. ap. and M. bovis.

In one embodiment, the diagnostic kit may be portable. The portablediagnostic kit may specifically suitable for field testing. Applicantsenvision that the present diagnostic kit may be used in a farm fieldsuch as a milk farm, where farmers/veterinarians may collect samples andrun the assay on the field (point of care assay) to identify earlystages of Johne's disease infection and to differentiate infected fromvaccinated mammals.

The kit may include a substrate. In one embodiment, the substrate may becoated with biomolecules such as antibodies, which are specificallybinding to the specific biomarkers as discussed above. The biomoleculesmay further possess a detectable label, such as a luminescent,fluorescent or radioactive label, and/or an affinity tag.

In one embodiment of the present invention, the substrate may be used asa sample holder. Exemplary substrates may include microtiter strips orplates. In one specific embodiment, a sample such as a diluted serum maybe pipetted into the wells of the microtiter plate or strip. A bindingbetween the biomarkers in the serum and the biomolecules takes place.The presence or absence of the specific biomarkers or a combination ofbiomarkers as discussed above may indicate the infection status of themammal.

The kit may further include a means of detection. The means of detectionmay include any detection method as discussed above. In one embodiment,the means of detection may be a spectroscopic technique, such as UV-Visor MS. In one specific embodiment, the means of detection may be ELISA.

In one embodiment, the kit may include standard data for specificbiomarker or a combination of biomarkers as discussed. One may comparethe test result of a mammalian sample with the standard data forspecific biomarker or a combination of biomarkers to determine theinfection status of the mammal For example, specific biomarkers or acombination of biomarkers may be visualized by a simple means ofdetection such as different colors. The detection result (e.g., showingone specific color) of a mammalian sample may be compared with thestandard data (e.g., different colors for different biomarkers) todetermine the infection status of the mammals.

In one embodiment, the kit may also be in the form of reagents (e.g.protein extract) that can be inoculated into animals to estimate thelevel of cell-mediate immunity (e.g. single intradermal comparative skintest, SICST). The reagents may include any of the biomarkers asdiscussed above. In one embodiment, the reagents may also include anygenetically engineered vaccines. Suitable genetically engineeredvaccines may include those Applicants previously proposed in PCT patentapplication US2014/02024.

The diagnostic kit may also include one or more of the following:instructions for use (detailing the method of the first aspect of theinvention); sample collection apparatus (such as a needle and syringe);a chart for interpretation of the results; an electronic readout system;software providing a database for accurate data management.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

EXAMPLES Example 1

Proteomic Analysis ofM. avium subsp. paratuberculosis VaccineCandidates.

Johne's disease (JD) is a worldwide health problem for dairy herds thatcarries a heavy economic burden for producing safe food. Infected cattlesuffer from chronic diarrhea, weight loss, low milk yield and low, butpersistent mortality (1). For the dairy industry alone, the economiclosses caused by JD are estimated to range between $200-$500 millionannually, in the USA alone (2, 3). Identifying protective vaccinecandidates against JD could be the cornerstone of controlling thiswidespread infection. In our group, we deciphered genomic informationavailable for M. ap to identify key gene regulators that could controlthe expression of large number of genes. Throughout the genome of M. apthere are 19 sigma factors that act as global gene regulators that couldcontribute to the ability of M. ap to grow in many environments (4).Through previous funding from USDA, we examined several M. ap sigma (σ )factors that were important for growth in murine macrophages. Usingtranscriptional profiling, we compared mid-log phase M. ap to M. ap thathad infected IFN-γ activated macrophages for 2 and 24 hours. Of the 19sigma factors monitored, 6 sigma factor transcripts were up-regulatedand one sigma factor transcript was down-regulated during the 24 hourtime frame. Of the up-regulated transcripts, the sigL transcript was theonly transcript up-regulated 2 hours after infection while sigH wasup-regulated at 24 hrs (5). SigL is implicated in cell membrane proteinbiosynthesis as well as virulence in M. tuberculosis (6) while SigH wasshown to be involved in combating the host intracellular responses suchas oxidative stress (7).

To assess the role of sigL and sigH in M. ap virulence, we replaced thetarget sigma factors gene coding regions with a hygromycin-resistantgene cassette in M. ap K-10 using a specialized transduction protocolthat was adapted for M. ap. Both genes were shown to be necessary for M.ap virulence in different stages of murine infection as detailedbefore(5, 8). Interestingly, the same mutants were shown to provideprotective immunity against challenge with the virulent strain of M. apwhen they were used as vaccine candidates in mice. To better analyzeproteins expressed in each mutant, we grow cultures of M. apΔsigL, M.apΔsigH mutants and the wild type parent strain, M. ap K10 to mid-logphase. All cultures were washed twice in PBS, resuspended in buffercocktail with endonuclease before proteomic analysis using nano-LiquidChromatography-Mass Spectroscopy-MS (nano-LC MS/MS) at the University ofWisconsin Biotechnology Center. From 3 biological replicates, a total of˜900 proteins were identified in this analysis comparing sigL and sigHmutant to M. ap K10 proteome.

Diagnostic Markers for JD-Vaccinated Animals.

A major problem in employing mass vaccination program for the control ofJD in dairy herds is the inability to differentiate between infected andvaccinated animals with the current vaccine (DIVA principal). Inaddition, vaccinated animals could not be differentiated from positivereaction to the infection with M. bovis, a significant health problemfor domesticated and wildlife animals. However, the DIVA principal andability to distinguish between M. bovis and JD vaccinated animals couldbe achieved in genetically engineered vaccines (such as live attenuatedvaccines based on sigL and sigH mutant) using a novel approach designedby the Applicant. In this approach, a simple blood test targetingproteins or sequences present in M. ap wild type strain and with lowerexpression level in the vaccine strain or even not encoded in the M.bovis genome would be developed. The target proteins include thefollowing list of genes that could be used for the sigL-based vaccines.

TABLE 1 M. ap proteins that are significantly over-expressed in the wildtype strain and not in the sigL-vaccine and could be used for sigL-DIVAtesting. Accession Fold Change Number Number (K10/sigH) Name/Function 1Q73SF4 1.75 hypothetical protein 2 Q73Y73 2.66 aldehyde dehydrogenase(NAD+) 3 Q73ZE6 2.13 nucleotide-sugar epimerase EpiA 4 Q73SL7 2.69hypothetical protein Mb0574c 5 Q73VK6 1.14 oxidoreductase 6 Q73XZ0 1.88antigen CFP2 7 Q740D1 4.71 peptide synthetase Nrp 8 Q73UE0 1.99 cutinase

TABLE 2 M. ap proteins that are significantly over-expressed in the wildtype strain and not in the sigH-vaccine and could be used for sigH-DIVAtesting. Fold Accession Change Number Number (WT/sigH) Name/Function 1Q73VL6 3.05 diguanylate cyclase (GGDEF) domain-containing protein 2Q73YW9 1.64 PE family protein, partial 3 Q741L4 1.88 hypotheticalprotein 4 Q744E5 2.67 ABC transporter ATPase 5 Q73YP5 2.47 Pup--proteinligase 6 Q73WE5 1.78 arginine decarboxylase 7 Q73U21 1.88 PE familyprotein PE17 8 Q73UH9 2.16 XRE family transcriptional regulator 9 Q741M52.11 nitroreductase 10 Q742F4 2.72 metallo-beta-lactamase 11 Q73SU6 2.473-ketoacyl-ACP reductase

In addition, another vaccine candidate is based on lipN mutant. In thiscase, epitopes that are different in the M. ap protein compared to thosein M. bovis will be the target for DIVA diagnostic test.

FIG. 1 shows the alignment plot of amino acids deduced from the proteinsequence in LipN of both M. paratuberculosis and M. bovis. Peptidesconserved in M. paratuberculosis sequence but absent from M. bovis wouldbe the target for developing the DIVA test.

Example 2 Prophetic Example

Objective: Develop Simple Assays to Differentiate Infected andVaccinated Animals (DIVA).

Taking advantage of the defined genetic mutation introduced in thecurrent vaccine candidates, we will develop a multiplex PCR-based assayto differentiate M. ap-infected from LAV-vaccinated animals. Inaddition, we will prepare whole cell lysates (WCL) from our LAVconstructs to be used for single intradermal comparative skin test(SICST) to differentiate M. bovis-infected from LAV-vaccinated animals.

Development of an efficient, safe and commercially viable vaccineagainst chronic infections is ordinarily arduous and prolonged. We havealready identified promising candidates that could significantly reduceM. ap infection in 2 different animal models. The long-term goal is todevelop these candidates into an easily administered, highly effectivevaccine to control JD in dairy operations. Our novel application ofadjuvant-supplemented LAV to create effective JD vaccines and thedevelopment of sensitive DIVA assays will further improve our chances tocommercialize this targeted vaccine.

Rationale.

An important hurdle in the use of a live attenuated vaccine (LAV) tocontrol JD is the ability to distinguish between vaccinated and infectedanimals. Infection with a wild type strain of M. ap or M. bovis willinterfere with the current standard tests for JD (PCR or ELISA) and forbovine tuberculosis (intradermal skin test). To overcome such hurdles,we designed a multiplex PCR-based assay using unique sequences presentin our LAV which will provide a simple, cost-effective assay todetermine if an animal has been vaccinated or is infected with virulentM. ap. This assay will take advantage of the reliability and speed ofamplification-based assays. To differentiate M. bovis-infected animalsform LAV-vaccinated animals, we will utilize the whole cell lysatesprepared from the LAV candidates to conduct a single intradermalcomparative skin test (SICST). We hypothesize that significantdifference in the antigenic composition of both LAV and M. bovis willallow us to develop a CMI-based assay. Our proteomic analysis providedus with an example of how the proteome of LAV construct can be highlydifferent from the wild type or M. bovis strains, even when a relativelysmall number of proteins were analyzed. In the proposed SICST, both M.bovis PPD and LAV-WCL will be inoculated to the same side of animal'sneck to compare the degree of induration for each preparation. A similartest using M. avium sensitin is already in use in several parts of theworld (Daniel et al., 2009; Gey van Pittius et al., 2012) whereinfection with the opportunistic M. avium is prevalent and need to bedifferentiated from infection with M. bovis. In our hands, goatsimmunized with LAV showed higher responses to M. ap PPD compared to M.bovis. Also, experimentally, we were able to show that PBMC cellsstimulated with M. ap whole cell lysate outperformed the M. ap PPD inthe standard IFN-γ release assay. Accordingly, in this part of theproject, we will use WCL from LAV instead of PPD, which could be missingkey stimulatory antigens.

Experimental Design.

Development of a DIVA using PCR. Our mutant strains are generated usinga previously reported method in which the target gene is replaced with ahygromycin-resistance cassette, which is then subsequently removed,leaving only a “scar” sequence in its place. Since this sequence isunique to our mutant strain, we can detect its presence using PCR. ThisPCR will be performed in multiplex with a second product amplified thatconsists of the original gene that was deleted in our vaccine strain(FIG. 2). Thus, a single PCR reaction with 3 primers will provide apositive result for both vaccinated animals and infected animals, withthe ability to differentiate them based on the size of the PCR product.This assay will be validated using fecal samples spiked with knownquantities of both virulent and LAV strains of M. ap. The amplificationtarget for this PCR will include fecal or saliva samples collected fromsuspected animals, similar to the sampling strategy we used before forassaying vaccine safety (preliminary results).

Evaluate the PCR assay using goat samples. Using fecal samples collectedfrom goats in Objective II (above), we will validate the DIVA assay byextracting DNA from fecal samples and subjecting it to our assay. ThePBS-vaccinated group will serve as a control for detecting only thechallenge strain. Validation of the developed test will be furtherevaluated on with samples collected from infected and vaccinated cows inPhase II of this project.

Development of SICST. We will prepare whole cell lysate from early, midand stationary phase cultures of our LAV candidates using standardprotocols (Al-Khodari et al., 2011; Xi et al., 2011). At 1 month postimmunization or 4 months post challenge (different times from regulartesting with Johnin), goats used for experiments in Objective II will beinoculated with 0.1 ml (100 μg) of Tuberculin PPD or LAV-WCL. At 72 hrspost inoculation, skin indurations will be measured using a digitalcaliper. Skin induration measurements will be compiled from all animalsand compared between groups. To start test interpretation, we can getguidance from similar tests performed on M. bovis infected animals(Bezos et al., 2010; Daniel et al., 2009). Any induration >5 mm of theWCL above the induration of the Tuberculin will be considered positivefor LAV immunization while the vice versa will be an indication ofinfection with M. bovis. To standardize the last part of this testinterpretation, we will have to have access to M. bovis infected goats.This part of the project will be performed as part of the testvalidation in Phase II of the project.

Expected results and alternative approaches. Data generated fromexperiments proposed here will develop easy to perform assays todistinguish vaccinated from M. ap or M. bovis-infected animals. Onepotential issue with the PCR-based assay is the requirement that theanimal is shedding bacteria or that bacterial DNA is present in thefeces. Since bacterial shedding in Johne's disease is intermittent, thisassay may not always be able to detect DNA from either strain. If thisapproach does not meet the needs of Johne's disease prevention programs,we will develop an ELISA-based assay. Since our LAV strains lack 1 ormore proteins due to deletion of the lipN and fabG2_2 genes, we coulddevelop an ELISA against these proteins.

In another embodiment of the invention, we will utilize proteomic dataon pgsN and/or pgsF to deduce new targets.

Finally, in another embodiment of the invention, the SICST might not besensitive to respond differentially to antigens from LAV or M. bovisinfection in some situations. In this case, we could increase theconcentration of the used antigens or use cytokine profiles associatedwith vaccinated animals as the DIVA assay. In this case, the test wouldbe completely evaluated in Phase II of this project when we can includeM. bovis infected animals for comparative analysis.

Example 3 Biomarkers for Early Diagnosis and Differentiation ofMycobacterial Infections

Johne's disease, caused by Mycobacterium avium subspeciesparatuberculosis (MAP) is a chronic gastroenteritis of ruminants.Although infection often occurs within the first few months of life,clinical signs do not appear until 2-5 years of age. Current diagnostictests, such as fecal culture and ELISA, have poor sensitivity fordetection of the sub-clinical phase of disease. Therefore, biomarkershave been increasingly investigated as a method for sub-clinicaldetection.

In this project, we set out to develop rapid assays (e.g. PCR or fieldskin test) for early detection of presence of Johne's disease and forthe differentiation of Johne's disease vaccinated vs. infected animals(with MAP or M. bovis). To speed up the project outcome, we capitalizedon ongoing vaccine study in goats (Capra hircus) and collectedPeripheral blood mononuclear cells (PBMC's) for transcriptionalprofiling followed by gene prediction for disease initiation andprogression.

The PBMC's have been shown to be a predictor of infection andinflammatory disease. The PBMC transcriptomes of the goats were profiledusing RNA-sequencing (RNA-Seq) to evaluate differential gene expressionbetween a subset of samples from either 30 days post-vaccination, 30days post-infection, or a naive, non-infected control group (3-4biological replicates per group). Preliminary results on differentialgene expression indicated the presence of 88 significantlydifferentially expressed genes out of 11,009 genes between goats at 30days post-infection and the naï, non-infected controls. The 30 dayspost-vaccination group had 720 out of 10,985 and 746 out of 11,099significantly differentially expressed genes compared to thenaïnon-infected control group and the 30 days post-infection group,respectively. However, preliminary evaluation of the expressed genesindicated a large number of genes with immunological and inflammatoryfunctions, including IL-18 binding protein, IFN-γ, IL-17A, and IL-22. Asa result of this inquiry, Table 3 summarizes selected genes/targetssuitable to use in the present invention.

TABLE 3 List of DNA markers that are derived from the host transcriptomeanalysis and can be used for early diagnosis of Johne's disease inruminants (e.g., cattle, goats, sheep and camels). Homolog in Bos taurusHomolog Locus in goats Symbol Protein Description (cows) description-Selected list of host (goat and cow) markers that can differentiateinfected from naïve animals. The loci of the up-regulated biomarkers ininfected animals are underlined and bolded. NW_005125111.1: unplaced N/AN/A 0-184 genomic scaffold NW_005101181.1: unplaced N/A N/A 1703-1858genomic scaffold NW_005101181.1: LOC102180841 XP_005701370.1 PREDICTED:XP_005199610 multidrug 168292-168418 multidrug resistance- resistance-associated associated protein 4-like protein 4-like isoform X1NC_022320.1: Non-coding N/A N/A 39973839-39974080 region NC_022297.1:IL-22 XP_005680263.1 interleukin 22 NP_001091849.1 interleukin 2244037534-44043184 NC_022296.1: Non-coding N/A N/A 81262820-81263390region NW_005101844.1: ABCC4 XP_005701761.1 PREDICTED: XP_010820300.1PREDICTED: 141791-142987 multidrug multidrug resistance- resistance-associated associated protein 4-like, protein 4 partial isoform X1NW_005101711.1: LOC102185556 XP_005701708.1 PREDICTED: XP_003585348.3PREDICTED: 48628-48757 multidrug multidrug resistance- resistance-associated associated protein 4-like protein 4 isoform X1NW_005132660.1: unplaced N/A N/A 0-240 genomic scaffold NW_005109943.1:unplaced N/A N/A 2-224 genomic scaffold NW_005149706.1: unplaced N/A N/A0-366 genomic scaffold NW_005153011.1: unplaced N/A N/A 2-407 genomicscaffold -Selected list of host (goat and cow) markers that candifferentiate Live attenuated vaccinated (LAV) from naïve animals. Theloci of the up-regulated biomarkers in vaccinated animals withlive-attenuated vaccines are underlined and bolded. NC_022296.1:Non-coding N/A N/A 32351255-32351413 region NC_022307.1: Non-coding N/AN/A 44045143-44403012 region NC_022295.1: Non-coding N/A N/A13176472-13182094 region NC_022321.1: Non-coding N/A N/A 6000551-6000875region NW_005126018.1: unplaced N/A N/A 16-203 genomic scaffoldNW_005101711.1: LOC102185556 XP_005701708.1 PREDICTED: XP_003585348.3PREDICTED: 48628-48757 multidrug multidrug resistance- resistance-associated associated protein 4-like protein 4 isoform X1NW_005101844.1: ABCC4 XP_005701761.1 PREDICTED: XP_010820300.1PREDICTED: 141790-142987 multidrug multidrug resistance- resistance-associated associated protein 4-like, protein 4 partial isoform X1NW_005101645.1: unplaced N/A N/A 16151-23647 genomic scaffold -Selectedlist of host (goat and cow) markers that can differentiateinactivated-vaccine immunized from naïve animals. The loci of theup-regulated biomarkers in vaccinated animals with inactivated vaccinesare underlined and bolded. NW_005125111.1: unplaced N/A N/A 0-184genomic scaffold NC_022320.1: Non-coding N/A N/A 39973839-39974080region NC_022303.1: Non-coding N/A N/A 46207878-46237242 regionNC_022296.1: Non-coding N/A N/A 81262820-81263390 region NW_005101711.1:LOC102185556 XP_005701708.1 PREDICTED: XP_003585348.3 PREDICTED:48628-48757 multidrug multidrug resistance- resistance- associatedassociated protein 4-like protein 4 isoform X1 NW_005102056.1:LOC102190036 XP_005701827.1 PREDICTED: NP_786982.1 tyrosine- 2049-9786tyrosine- protein protein phosphatase phosphatase non-receptornon-receptor type type substrate 1 substrate 1- precursor like, partialNC_022309.1: — XP_005691363.1 PREDICTED: NP_001077247.1 protein40520580-40588889 protein FAM198B FAM198B NW_005101931.1: LOC102180487XP_005701808.1 PREDICTED: NP_001069925.2 uncharacterized 48185-54192interferon protein alpha- LOC617420 inducible protein 27- like protein2- like NW_005164924.1: unplaced N/A N/A 1-636 genomic scaffold

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We claim:
 1. A method for diagnosis of mycobacterial infection in amammal, the method comprising the steps of a) obtaining a sample fromthe mammal; b) testing the sample for the concentration level of atleast one mycobacterial-specific biomarker and comparing the level ofthe biomarker against the level detected in an uninfected mammaliansample; and c) determining the infection status of the mammal.
 2. Themethod of claim 1, wherein the method is used for early diagnosis anddetection of mycobacterial infection in a mammal.
 3. The method of claim1, wherein the testing is via ELISA assay for antibodies formed againstthe biomarker.
 4. The method of claim 1, wherein the sample is a bloodsample.
 5. The method of claim 1, wherein the mammal is selected fromthe group consisting of bubaline, elephantine, musteline, pardine,phocine, rhinocerine, caprine, hircine, leonine, leporine, lupine,lyncine, murine, rusine, tigrine, ursine, vulpine, zebrine,vespertilionine, porcine, bovine, equine, swine, elaphine, ovine,caprine, camelidae, feline, cervine, primate, human and canine mammals.6. The method of claim 1 wherein the mammal is selected from the groupof pig, cow, human, rodent, sheep, goat and deer.
 7. The method of claim1, wherein the mammal is selected from the group consisting of cow,sheep and goat.
 8. The method of claim 1, wherein the mammal is a cow.9. The method of claim 1, wherein the mycobacterial-specific biomarkercomprises at least one member selected from the group consisting of genesequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZ0, Q740D1 andQ73UE0 and expression products derived thereof.
 10. The method of claim1, wherein the mycobacterial-specific biomarker comprises at least onemember selected from the group consisting of gene sequences Q73VL6,Q73YW9, Q741 L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741 M5,Q742F4, and Q73SU6 and expression products derived thereof.
 11. Themethod of claim 1, wherein the mycobacterial-specific biomarkercomprises at least one member selected from Table
 3. 12. The method ofclaim 1, wherein the biomarker comprises a protein having at least 50%,60%, 70%, 80%, or 90% of the amino acids of M. paratuberculosis whichare not conserved in M. bovis as exemplified by the amino acid sequencedifference between M. paratuberculosis and M. bovis in FIG.
 1. 13. Amethod for differentiating a vaccinated mammal from a non-vaccinatedmammal or from an infected mammals, the method comprising the steps ofa) obtaining a sample from the test mammal; b) testing the sample forthe concentration level of at least one of mycobacterial specificmarkers and comparing the level of the markers with that detected in anuninfected animal; and c) determining the status of the mammal.
 14. Themethod of claim 13, wherein the mammals are vaccinated with amycobacterium mutant vaccine.
 15. The method of claim 14, wherein themycobacterium mutant vaccine comprises at least one mutation in at leastone gene sequence encoding global gene regulators (GGRs) selected fromthe group consisting of sigH, sigL and LipN.
 16. The method of claim 13,wherein the marker comprises at least one member selected from the groupconsisting of Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1 andQ73UE0 and expression products derived thereof.
 17. The method of claim13, wherein the marker comprises at least one member selected from thegroup consisting of Q73VL6, Q73YW9, Q741 L4, Q744E5, Q73YP5, Q73WE5,Q73U21, Q73UH9, Q741 M5, Q742F4, and Q73SU6 and expression productsderived thereof.
 18. The method of claim 13, wherein the markercomprises at least one member selected from Table
 3. 19. The method ofclaim 13, wherein the marker comprises a protein having at least 50%,60%, 70%, 80%, or 90% of the amino acid sequence of M. paratuberculosiswhich is not conserved in M. bovis, as exemplified by the amino aciddifference between M. paratuberculosis and M. bovis in FIG.
 1. 20. Amethod for differentiating a M. ap infected mammal from a M. bovisinfected mammal, the method comprising the steps of a) obtaining asample from the test mammal; b) testing the sample for the concentrationlevel of at least one of mycobacterial-specific markers and comparingthe level of the markers with that detected in a M. bovis infectedmammal; and c) determining the status of the mammal.
 21. The method ofclaim 20, wherein the mycobacterial-specific markers comprise a proteinhaving at least 50%, 60%, 70%, 80%, or 90% of the amino acid sequence ofM. paratuberculosis which is not conserved in M. bovis.
 22. A set ofbiomarkers for early diagnosis and differentiation of mycobacterialinfection, the biomarkers comprising: at least one member selected fromthe group consisting of Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZO,Q740D1 and Q73UE0 and expression products derived thereof.
 23. The setof biomarkers in claim 22, wherein the biomarkers comprising at leasttwo members selected from the group consisting of Q73SF4, Q73Y73,Q73ZE6, Q73SL7, Q73VK6, Q73XZO, Q740D1 and Q73UE0 and expressionproducts derived thereof.
 24. A set of biomarkers for early diagnosis ordifferentiation of mycobacterial infection, the biomarkers comprising:at least one member selected from the group consisting of Q73VL6,Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4,and Q73SU6 and encoded genes or expression products derived thereof. 25.The set of biomarkers in claim 24, wherein the biomarkers comprising atleast two members selected from the group consisting of Q73VL6, Q73YW9,Q741 L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, andQ73SU6 and encoded genes or expression products derived thereof.
 26. Aset of biomarkers for early diagnosis or differentiation ofmycobacterial infection, the biomarkers comprising a protein having atleast 50%, 60%, 70%, 80%, 90%, 95% or 100% of the amino acid sequence ofM. paratuberculosis which is not conserved in M. bovis.
 27. A set ofbiomarkers for early diagnosis or differentiation of mycobacterialinfection, the biomarkers comprising at least one member selected fromTable 3.